Microfluidic devices for fluid manipulation and analysis

ABSTRACT

The present invention relates to microfluidic devices and methods for manipulating and analyzing fluid samples. The disclosed microfluidic devices utilize a plurality of microfluidic channels, inlets, valves, filter, pumps, liquid barriers and other elements arranged in various configurations to manipulate the flow of a fluid sample in order to prepare such sample for analysis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.13/225,280, filed Sep. 2, 2011 (now allowed), which application is adivisional of U.S. patent application Ser. No. 12/685,582, filed Jan.11, 2010 (now issued as U.S. Pat. No. 8,318,109); which application is adivisional of U.S. patent application Ser. No. 12/182,434, filed Jul.30, 2008 (now abandoned); which application is a continuation of U.S.patent application Ser. No. 10/870,717, filed Jun. 17, 2004 (now issuedas U.S. Pat. No. 7,419,638); which application is a continuation-in-partof U.S. patent application Ser. No. 10/757,767, filed Jan. 14, 2004 (nowabandoned); which application claims the benefit of U.S. ProvisionalPatent Application Nos. 60/439,825, filed Jan. 14, 2003, and 60/441,873,filed Jan. 21, 2003, all of which applications are hereby incorporatedby reference in their entireties.

BACKGROUND

1. Technical Field

The present invention relates generally to microfluidic devices andanalysis methods, and, more particularly, to microfluidic devices andmethods for the manipulation and analysis of fluid samples.

2. Description of the Related Art

Microfluidic devices have become popular in recent years for performinganalytical testing. Using tools developed by the semiconductor industryto miniaturize electronics, it has become possible to fabricateintricate fluid systems which can be inexpensively mass produced.Systems have been developed to perform a variety of analyticaltechniques for the acquisition and processing of information.

The ability to perform analyses microfluidically provides substantialadvantages of throughput, reagent consumption, and automatability.Another advantage of microfluidic systems is the ability to integrate aplurality of different operations in a single “lap-on-a-chip” device forperforming processing of reactants for analysis and/or synthesis.

Microfluidic devices may be constructed in a multi-layer laminatedstructure wherein each layer has channels and structures fabricated froma laminate material to form microscale voids or channels where fluidsflow. A microscale or microfluidic channel is generally defined as afluid passage which has at least one internal cross-sectional dimensionthat is less than 500 μm and typically between about 0.1 μm and about500 μm.

U.S. Pat. No. 5,716,852, which patent is hereby incorporated byreference in its entirety, is an example of a microfluidic device. The'852 patent teaches a microfluidic system for detecting the presence ofanalyte particles in a sample stream using a laminar flow channel havingat least two input channels which provide an indicator stream and asample stream, where the laminar flow channel has a depth sufficientlysmall to allow laminar flow of the streams and length sufficient toallow diffusion of particles of the analyte into the indicator stream toform a detection area, and having an outlet out of the channel to form asingle mixed stream. This device, which is known as a T-Sensor, allowsthe movement of different fluidic layers next to each other within achannel without mixing other than by diffusion. A sample stream, such aswhole blood, a receptor stream, such as an indicator solution, and areference stream, which may be a known analyte standard, are introducedinto a common microfluidic channel within the T-Sensor, and the streamsflow next to each other until they exit the channel. Smaller particles,such as ions or small proteins, diffuse rapidly across the fluidboundaries, whereas larger molecules diffuse more slowly. Largeparticles, such as blood cells, show no significant diffusion within thetime the two flow streams are in contact.

Typically, microfluidic systems require some type of external fluidicdriver to function, such as piezoelectric pumps, micro-syringe pumps,electroosmotic pumps, and the like. However, in U.S. patent applicationSer. No. 09/684,094, which application is assigned to the assignee ofthe present invention and is hereby incorporated by reference in itsentirety, microfluidic systems are described which are completely drivenby inherently available internal forces such as gravity, hydrostaticpressure, capillary force, absorption by porous material or chemicallyinduced pressures or vacuums.

In addition, many different types of valves for use in controllingfluids in microscale devices have been developed. For example, U.S. Pat.No. 6,432,212 describes one-way valves for use in laminated microfluidicstructures, U.S. Pat. No. 6,581,899 describes ball bearing valves foruse in laminated microfluidic structures, and U.S. patent applicationSer. No. 10/114,890, which application is assigned to the assignee ofthe present invention, describes a pneumatic valve interface, also knownas a zero dead volume valve, for use in laminated microfluidicstructures. The foregoing patents and patent applications are herebyincorporated by reference in their entirety.

Although there have been many advances in the field, there remains aneed for new and improved microfluidic devices for manipulating andanalyzing fluid samples. The present invention addresses these needs andprovides further related advantages.

BRIEF SUMMARY

In brief, the present invention relates to microfluidic devices andmethods for manipulating and analyzing fluid samples. The disclosedmicrofluidic devices utilize a plurality of microfluidic channels,inlets, valves, filters, pumps, liquid barriers and other elementsarranged in various configurations to manipulate the flow of a fluidsample in order to prepare such sample for analysis. Analysis of thesample may then be performed by any means known in the art. For example,as disclosed herein, microfluidic devices of the present invention maybe used to facilitate the reaction of a blood sample with one or morereagents as part of a blood typing assay.

In one embodiment, a microfluidic device for analyzing a liquid sampleis provided that comprises (a) a microfluidic channel having a first endand a second end, (b) a sample inlet fluidly connected to the first endof the microfluidic channel for receiving the liquid sample, (c) afilter interposed between the sample inlet and the first end of themicrofluidic channel, wherein the filter removes selected particles fromthe liquid sample, (d) a bellows pump fluidly connected to the secondend of the microfluidic channel, and (e) a liquid barrier interposedbetween the bellows pump and the second end of the microfluidic channel,wherein the liquid barrier is gas permeable and liquid impermeable.

In further embodiments, the bellows may comprise a vent hole, the filtermay comprise a membrane, or the microfluidic device may further comprise(a) a first check valve interposed between the bellows pump and theliquid barrier, wherein the first check valve permits fluid flow towardsthe bellows pump, and (b) a second check valve fluidly connected to thebellows pump, wherein the second check valve permits fluid flow awayfrom the bellows pump.

In another embodiment, a microfluidic device for analyzing a liquidsample is provided that comprises (a) a first microfluidic channelhaving a first end and a second end, (b) a sample inlet fluidlyconnected to the first end of the first microfluidic channel forreceiving the liquid sample, (c) an active valve interposed between thesample inlet and the first end of the first microfluidic channel, (d) ameans for actuating the active valve, (e) a first bellows pump fluidlyconnected to the second end of the first microfluidic channel, (f) aliquid barrier interposed between the first bellows pump and the secondend of the first microfluidic channel, wherein the liquid barrier is gaspermeable and liquid impermeable, (g) a second microfluidic channelhaving a first end and a second end, wherein the first end is fluidlyconnected to the first microfluidic channel at a location adjacent tothe active valve, (h) a passive valve interposed between the first endof the second microfluidic channel and the first microfluidic channel,wherein the passive valve is open when the fluid pressure in the firstmicrofluidic channel is greater than the fluid pressure in the secondmicrofluidic channel, and (i) a sample reservoir fluidly connected tothe second end of the second microfluidic channel.

In further embodiments, the first bellows pump may comprise a vent hole,the means for actuating the active valve may comprise a second bellowspump and/or the sample reservoir may comprise a vent hole.

In another embodiment, a microfluidic device for analyzing a liquidsample is provided that comprises (a) first and second microfluidicchannels, each having a first end and a second end, (b) a sample inletfluidly connected to the first end of the first microfluidic channel forreceiving the liquid sample, (c) a first bellows pump fluidly connectedto, and interposed between, the second end of the first microfluidicchannel and the first end of the second microfluidic channel, (d) asecond bellows pump fluidly connected to the second end of the secondmicrofluidic channel, wherein the second bellows pump has a fluidoutlet, (e) a first check valve interposed between the sample inlet andthe first end of the first microfluidic channel, wherein the first checkvalve permits fluid flow towards the first microfluidic channel, (f) asecond check valve interposed between the second end of the firstmicrofluidic channel and the first bellows pump, wherein the secondcheck valve permits fluid flow towards the first bellows pump, (g) athird check valve interposed between the first bellows pump and thefirst end of the second microfluidic channel, wherein the third checkvalve permits fluid flow towards the second microfluidic channel, and(h) a fourth check valve interposed between the second end of the secondmicrofluidic channel and the second bellows pump, wherein the fourthcheck valve permits fluid flow towards the second bellows pump.

In another embodiment, a microfluidic device for analyzing a liquidsample is provided that comprises (a) a first microfluidic channelhaving a first end and a second end, (b) a sample inlet fluidlyconnected to the first end of the first microfluidic channel forreceiving the liquid sample, (c) a first reagent inlet fluidly connectedto the first end of the first microfluidic channel for receiving a firstreagent, (d) a bellows pump fluidly connected to the second end of thefirst microfluidic channel, and (e) a first liquid barrier interposedbetween the bellows pump and the second end of the first microfluidicchannel, wherein the liquid barrier is gas permeable and liquidimpermeable.

In further embodiments, the bellows pump may comprise a vent hole or themicrofluidic device may further comprise a check valve fluidly connectedto the bellows pump, wherein the check valve permits fluid flow awayfrom the bellows pump.

In another further embodiment, the microfluidic device further comprises(a) a second microfluidic channel having a first end, fluidly connectedto the sample inlet, and a second end, fluidly connected to the bellowspump, (b) a second reagent inlet fluidly connected to the first end ofthe second microfluidic channel for receiving a second reagent, and (c)a second liquid barrier interposed between the bellows pump and thesecond end of the second microfluidic channel, wherein the second liquidbarrier is gas permeable and liquid impermeable.

In yet another further embodiment, the microfluidic device furthercomprises (a) a third microfluidic channel having a first end, fluidlyconnected to the sample inlet, and a second end, fluidly connected tothe bellows pump, (b) a third reagent inlet fluidly connected to thefirst end of the third microfluidic channel for receiving a thirdreagent, and (c) a third liquid barrier interposed between the bellowspump and the second end of the third microfluidic channel, wherein thethird liquid barrier is gas permeable and liquid impermeable.

In one alternate embodiment of the foregoing, the first reagent inletcomprises a first blister pouch containing the first reagent, the secondreagent inlet comprises a second blister pouch containing the secondreagent, and the third reagent inlet comprises a third blister pouchcontaining the third reagent.

In another embodiment, a microfluidic device for analyzing a liquidsample is provided that comprises (a) a first microfluidic channelhaving a first end and a second end, (b) a sample inlet fluidlyconnected to the first end of the first microfluidic channel forreceiving the liquid sample, (c) a first dried reagent zone, comprisinga first reagent printed thereon, fluidly connected to the first end ofthe first microfluidic channel, (d) a bellows pump fluidly connected tothe second end of the first microfluidic channel, and (e) a first liquidbarrier interposed between the bellows pump and the second end of thefirst microfluidic channel, wherein the liquid barrier is gas permeableand liquid impermeable.

In further embodiments, the bellows pump may comprise a vent hole or themicrofluidic device may further comprise a check valve fluidly connectedto the bellows pump, wherein the check valve permits fluid flow awayfrom the bellows pump.

In another further embodiment, the microfluidic device further comprises(a) a second microfluidic channel having a first end, fluidly connectedto the sample inlet, and a second end, fluidly connected to the bellowspump, (b) a second dried reagent zone, comprising a second reagentprinted thereon, fluidly connected to the first end of the secondmicrofluidic channel, and (c) a second liquid barrier interposed betweenthe bellows pump and the second end of the second microfluidic channel,wherein the second liquid barrier is gas permeable and liquidimpermeable.

In yet another further embodiment, the microfluidic device furthercomprises (a) a third microfluidic channel having a first end, fluidlyconnected to the sample inlet, and a second end, fluidly connected tothe bellows pump, (b) a third dried reagent zone, comprising a thirdreagent printed thereon, fluidly connected to the first end of the thirdmicrofluidic channel, and (c) a third liquid barrier interposed betweenthe bellows pump and the second end of the third microfluidic channel,wherein the third liquid barrier is gas permeable and liquidimpermeable.

In a more specific embodiment, the liquid sample comprises a bloodsample, the first reagent comprises antibody-A, the second reagentcomprises antibody-B, and the third reagent comprises antibody-D.

In yet a further embodiment, the microfluidic device further comprises ahydrating buffer inlet, fluidly connected to the first, second and thirddried reagent zones and to the first ends of the first, second and thirdmicrofluidic channels, for receiving a hydrating buffer. In an alternateembodiment, the hydrating buffer inlet comprises a hydrating bufferblister pouch containing the hydrating buffer.

These and other aspects of the invention will be apparent upon referenceto the attached figures and following detailed description.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIGS. 1A-1C are a series of cross-sectional views illustrating theoperation of a first embodiment of a microfluidic device in accordancewith aspects of the present invention.

FIGS. 2A-2C are a series of cross-sectional views illustrating theoperation of a second embodiment of a microfluidic device in accordancewith aspects of the present invention.

FIGS. 3A-3F are a series of cross-sectional views illustrating theoperation of a third embodiment of a microfluidic device in accordancewith aspects of the present invention.

FIGS. 4A-4E are a series of cross-sectional views illustrating theoperation of a fourth embodiment of a microfluidic device in accordancewith aspects of the present invention.

FIGS. 5A-5C are a series of cross-sectional views illustrating theoperation of a fifth embodiment of a microfluidic device in accordancewith aspects of the present invention.

FIGS. 6A-6F are schematic illustrations of blood typing cards inaccordance with aspects of the present invention.

FIGS. 7A-7C are a series of cross-sectional views illustrating theoperation of a sixth embodiment of a microfluidic device in accordancewith aspects of the present invention.

FIGS. 8A-8C are a series of cross-sectional views illustrating theoperation of a seventh embodiment of a microfluidic device in accordancewith aspects of the present invention.

DETAILED DESCRIPTION

As noted previously, the present invention relates to microfluidicdevices and methods utilizing a plurality of microfluidic channels,inlets, valves, membranes, pumps, liquid barriers and other elementsarranged in various configurations to manipulate the flow of a fluidsample in order to prepare such sample for analysis and to analyze thefluid sample. In the following description, certain specific embodimentsof the present devices and methods are set forth, however, personsskilled in the art will understand that the various embodiments andelements described below may be combined or modified without deviatingfrom the spirit and scope of the invention.

FIGS. 1A-1C are a series of cross-sectional views of the device 110illustrating the operation of a first embodiment of the invention. Asshown in FIG. 1A, microfluidic device 110 comprises a microfluidicchannel 120 having a first end 122 and a second end 124. As illustrated,device 110 is in the form of a cartridge, however, the form of device110 is not essential to the present invention, and persons of ordinaryskill in the art can readily select a suitable form for a givenapplication. The microfluidic devices of the present invention, such asdevice 110, may be constructed from a material, such as transparentplastic, mylar or latex, using a method such as injection molding orlamination.

As further shown in FIG. 1A, device 110 comprises a sample inlet 130fluidly connected to first end 122 of microfluidic channel 120 forreceiving a liquid sample and a filter 140 interposed between sampleinlet 130 and first end 122 of microfluidic channel 120. Filter 140 iscapable of removing selected particles, such as white blood cells, redblood cells, polymeric beads, such as polystyrene or latex with sizesfrom 1-100 microns, and bacteria cells, such as E. coli, from the liquidsample, and may comprise a membrane (as illustrated). A bellows pump 150having a vent hole 152 is fluidly connected to second end 124 ofmicrofluidic channel 120 and a liquid barrier 160 is interposed betweenbellows pump 150 and second end 124 of microfluidic channel 120. Liquidbarrier 160 is a gas permeable and fluid impermeable membrane.

During operation, a liquid sample in placed into sample inlet 130 (asshown in FIG. 1B), bellows pump 150 is depressed, either manually by auser or mechanically by an external device, vent hole 152 issubstantially sealed, such as by covering vent hole 152, and bellowspump 150 is then released. During depression of bellows pump 150, venthole 152 remains uncovered so that fluid in bellows pump 150 may beexpelled through vent hold 152. Upon release of bellows pump 150, anegative fluid pressure is created in microfluidic channel 120 and theliquid sample is drawn through filter 140 into, and through,microfluidic channel 120 to the liquid barrier 160 (as shown in FIG.1C).

As further shown in FIG. 1A, microfluidic channel 120 may comprise oneor more optical viewing area(s) 170. Optical viewing area(s) 170 enablevisual verification by a user that the liquid sample is flowing throughmicrofluidic channel 120.

FIGS. 2A-2C are a series of cross-sectional views of the device 210illustrating the operation of a second embodiment of the invention.Microfluidic device 210 illustrated in FIG. 2A is similar to device 110of FIG. 1A and comprises a microfluidic channel 220 having a first end222 and a second end 224, a sample inlet 230 fluidly connected to firstend 222 of microfluidic channel 220 for receiving a liquid sample, afilter 240 interposed between sample inlet 230 and first end 222 ofmicrofluidic channel 220, a bellows pump 250 fluidly connected to secondend 224 of microfluidic channel 220 and a liquid barrier 260 interposedbetween bellows pump 250 and second end 224 of microfluidic channel 220.

Rather than providing a vent hole in bellows pump 250 as in FIG. 1A,device 210 utilizes first and a second check valves, 254 and 256,respectively, to prevent the fluid in bellows pump 250 from beingexpelled into microfluidic channel 220 during depression of bellows pump250. Check valves, also known as one-way valves, permit fluid flow inone direction only. Exemplary check valves for use in microfluidicstructures are described in U.S. Pat. No. 6,431,212, which is herebyincorporated by reference in its entirety. First check valve 254 isinterposed between bellows pump 250 and liquid barrier 224 and permitsfluid flow towards bellows pump 250. Second check valve 256 is fluidlyconnected to bellows pump 250 and permits fluid flow away from thebellows pump (for example, by venting to the atmosphere).

During operation, a liquid sample is placed into sample inlet 230 (asshown in FIG. 2B), bellows pump 250 is depressed, either manually by auser or mechanically by an external device, and, then, bellows pump 250is released. During depression of bellows pump 250, first check valve254 remains closed and prevents fluid flow from bellows chamber 250 intomicrofluidic channel 220; second check valve 256 opens and expels thefluid displaced from bellows pump 250. Upon release of bellows pump 250,a negative fluid pressure is created, first check valve 254 opens andpermits fluid flow from microfluidic channel 220 into bellows pump 250,second check valve 256 closes and prevents fluid flow into bellows pump250 from, for example, the atmosphere, and the liquid sample is drawnthrough filter 240 into, and through, microfluidic channel 220 to liquidbarrier 260 (as shown in FIG. 2C).

In addition, similar to FIG. 1A, microfluidic channel 220 may optionallycomprise one or more optical viewing area(s) 270 to enable visualverification by a user that the liquid sample is flowing throughmicrofluidic channel 220.

FIGS. 3A-3F are a series of cross-sectional views illustrating theoperation of a third embodiment of the present invention. As shown inFIG. 3A, microfluidic device 310 comprises a first microfluidic channel320 having a first end 322 and a second end 324. A sample inlet 330 isfluidly connected to first end 322 of first microfluidic channel 320 forreceiving a liquid sample. A first bellows pump 350, having a vent hole352, is fluidly connected to second end 324 of first microfluidicchannel 320. Liquid barrier 360 is interposed between first bellows pump350 and second end 324 of microfluidic channel 320. As in FIGS. 1A and2A, the liquid barrier 360 is a gas permeable and liquid impermeablemembrane.

Furthermore, device 310 comprises an on/off active valve 370 interposedbetween sample inlet 330 and first end 322 of first microfluidic channel320 and a means 372 for actuating active valve 370. As illustrated,means 372 comprise a second bellows pump 372, however, persons ofordinary skill in the art can readily select an alternative and suitablemeans for applying manual or fluidic pressure to actuate active valve370. Device 310 also comprises a second microfluidic channel 380 havinga first end 382 and a second end 384. As shown, first end 382 of secondmicrofluidic channel 380 is fluidly connected to first microfluidicchannel 320 at a location adjacent to active valve 370 and second end384 of second microfluidic channel 380 is fluidly connected to a samplereservoir 390 having a vent hole 392. A passive valve 375 is interposedbetween first end 382 of second microfluidic channel 380 and firstmicrofluidic channel 320. Passive valve 375 is designed to be open whenthe fluid pressure in first microfluidic channel 320 is greater than thefluid pressure in second microfluidic channel 380. Exemplary passivevalves, also known as zero dead volume valves, for use in microfluidicstructures are described in U.S. patent application Ser. No. 10/114,890,which application is assigned to the assignee of the present inventionand is hereby incorporated by reference in its entirety.

During initial operation, a liquid sample is placed into sample inlet330 (as shown in FIG. 3B), first bellows pump 350 is depressed, eithermanually by a user or mechanically by an external device, vent hole 352is covered and, then, first bellows pump 350 is released. Duringdepression of first bellows pump 350, vent hole 352 remains uncovered sothat fluid in first bellows pump 350 may be expelled through vent hold352. Upon release of first bellows pump 350, a negative fluid pressureis created in microfluidic channel 320 and the liquid sample is drawnthrough active valve 370 and into, and through, microfluidic channel 320to liquid barrier 360 (as shown in FIG. 3C). During this initialdepression and release of first bellows pump 350, the fluid pressure infirst microfluidic channel 320 is less than the fluid pressure in secondmicrofluidic channel 380, thus passive valve 375 is closed and theliquid sample is prevented from flowing into second microfluidic channel380.

During the next stage of operation, shown in FIG. 3D, vent hole 352 iscovered, second bellows pump 372 is depressed, thereby actuating (i.e.,closing) active valve 370, and, then, first bellows pump 350 isdepressed, thereby creating a positive fluid pressure in firstmicrofluidic channel 320. As a result, the fluid pressure in firstmicrofluidic channel 320 rises above (i.e., is greater than) the fluidpressure in second microfluidic channel 380, passive valve 375 opens,and the liquid sample is pushed from first microfluidic channel 320 intosecond microfluidic channel 380.

During an additional stage of operation, the foregoing two steps arerepeated to draw an additional portion of the liquid sample into firstmicrofluidic channel 320, and, then, push the additional portion of theliquid sample into second microfluidic channel 380, thereby pushing thefirst portion of the liquid sample already in second microfluidicchannel 380 into sample reservoir 390. Depending on the amount of liquidsample and the size of sample reservoir 390, the foregoing additionalstage of operation may be repeated a number of times.

As further shown in FIGS. 3A-3F, more than one of the microfluidicchannel, pump and valve assemblies of the present invention may bedisposed in a single microfluidic device. In this way, a number of fluidmanipulations and analysis may be performed contemporaneously.

FIGS. 4A-4E are a series of cross-sectional views illustrating theoperation of a fourth embodiment of the invention. As shown in FIG. 4A,microfluidic device 410 comprises a first microfluidic channel 420having a first end 422 and a second end 424, a second microfluidicchannel 430 having a first end 432 and a second end 434, and a thirdmicrofluidic channel 440 having a first end 442 and a second end 444. Asample inlet 415, for receiving a liquid sample, is fluidly connectedto, both, first end 422 of first microfluidic channel 420 and second end444 of third microfluidic channel 440. A first bellows pump 450 isfluidly connected to, and interposed between, second end 424 of firstmicrofluidic channel 420 and first end 432 of second microfluidicchannel 430 and a second bellows pump 460 is fluidly connected to, andinterposed between, second end 434 of second microfluidic channel 430and first end 442 of third microfluidic channel 440.

As shown, device 410 also comprises a plurality of check valves. A firstcheck valve 470 is interposed between sample inlet 415 and first end 422of first microfluidic channel 420, and permits fluid flow towards firstmicrofluidic channel 420. A second check valve 472 is interposed betweensecond end 424 of first microfluidic channel 420 and first bellows pump450, and permits fluid flow towards first bellows pump 450. A thirdcheck valve 474 is interposed between first bellows pump 450 and firstend 432 of second microfluidic channel 430, and permits fluid flowtowards second microfluidic channel 430. A fourth check valve 476 isinterposed between second end 434 of second microfluidic channel 430 andsecond bellows pump 460, and permits fluid flow towards second bellowspump 460. A fifth check valve 478 is interposed between second bellowspump 460 and first end 442 of third microfluidic channel 440, andpermits fluid flow towards third microfluidic channel 440. A sixth checkvalve 480 is interposed between second end 444 of third microfluidicchannel 440 and sample inlet 415, and permits fluid flow towards sampleinlet 415. As in FIG. 2A, first, second, third, fourth, fifth and sixthcheck valves, 470, 472, 474, 476, 478 and 480, permit fluid flow in onedirection only (as noted by the arrows in FIG. 4A). As noted before,exemplary check valves for use in microfluidic structures are describedin U.S. Pat. No. 6,431,212.

During operation, a liquid sample in placed into sample inlet 415 (asshown in FIG. 4B) and first and second bellows pumps 450 and 460 arealternately, sequentially and/or repeatedly depressed and released,either manually by a user or mechanically by an external device, to drawand push the liquid sample through first, second and third microfluidicchannels 420, 430 and 440 (as shown in FIGS. 4C through 4E). Duringthese series of depressions and releases, first, second, third, fourth,fifth and sixth check valves, 470, 472, 474, 476, 478 and 480, ensurethat the liquid sample flows in one continuous direction throughmicrofluidic device 410.

In variations of this fourth embodiment, rather than being fluidlyconnected to a third microfluidic channel 440, which is fluidlyconnected to sample inlet 415 to form a fluidic loop, one or more fluidoutlet(s) of second bellows pump 460 may be fluidly connected to one ormore microfluidic channel(s), which are, in turn, fluidly connected toone or more additional microfluidic channel(s), bellows pumps and checkvalves. In this way, a person of ordinary skill in the art willappreciate that a series of check valves and bellows pumps may beassembled and utilized in a multitude of different configurations tomove a liquid sample through a network of microfluidic channels.

FIGS. 5A-5C are a series of cross-sectional views of a microfluidicdevice 510 illustrating the operation of a fifth embodiment of theinvention. Microfluidic device 510 illustrated in FIG. 5A comprises afirst microfluidic channel 520 having a first end 522 and a second end524, a second microfluidic channel 530 having a first end 532 and asecond end 534, and a third microfluidic channel 540 having a first end542 and a second end 544. Sample inlet 518 is fluidly connected to firstends 522, 532 and 542 of first, second and third microfluidic channels520, 530 and 540.

Device 510 further comprises a first reagent inlet 512 for receiving afirst reagent, a second reagent inlet 514 for receiving a second reagentand a third reagent inlet 516 for receiving a third reagent. Inalternate embodiments, the first, second and third reagents may beloaded during the manufacture of device 510 and first, second and thirdreagent inlets 512, 514 and 516 may comprise, for example, first, secondand third blister pouches (not shown) containing the first, second andthird reagents. Such blister pouches are adapted to burst, or otherwiserelease the first, second and third reagents into device 510, uponactuation, such as, for example, depression of the blister poucheseither manually by a user or mechanically by an external device.

As illustrated, each of the first, second and third reagent inlets 512,514 and 516 are fluidly connected to first ends 522, 532 and 542 offirst, second and third microfluidic channels 520, 530 and 540. Bellowspump 550 is fluidly connected to second ends 524, 534 and 544 of first,second and third microfluidic channels 520, 530 and 540, and first,second and third liquid barriers 526, 536 and 546 are interposed betweenbellows pump 550 and second ends 524, 534 and 544 of first, second andthird microfluidic channels 520, 530 and 540. As in FIGS. 1A, 2A and 3A,first, second and third liquid barriers 526, 536 and 546 are gaspermeable and liquid impermeable membranes.

As shown, bellows pump 550 is fluidly connected to a check valve 552,which permits fluid flow away from bellows pump 550. Alternatively, thebellows pump may comprise a vent hole as in the embodiments of FIGS. 1Aand 3A.

During operation, a liquid sample in placed into sample inlet 518, afirst reagent in placed into first reagent inlet 512, a second reagentis placed into second reagent inlet 514 and a third reagent is placedthird reagent inlet 516 as shown in FIG. 5B. (In the alternateembodiment, wherein first, second and third reagent inlets 512, 514 and516 comprise blister pouches containing the first, second and thirdreagents, operation is commenced by placing a liquid sample into sampleinlet 518 and manually actuating the blister pouches to release thefirst, second and third reagents). Bellows pump 550 is then depressed,either manually by a user or mechanically by an external device, and,then, bellows pump 550 is released. During depression of bellows pump550, check valve 552, or a vent hole (not shown), prevents fluid flowfrom bellows pump 550 into first, second and third microfluidic channels520, 530 and 540. Upon release of bellows pump 550, a negative fluidpressure is created in first, second and third microfluidic channels520, 530 and 540 and the liquid sample, the first reagent, the secondreagent and the third reagent are drawn into, and through, first, secondand third microfluidic channels 520, 530 and 540 to first, second andthird liquid barriers 526, 536 and 546 (as shown in FIG. 5C). Duringthis process, mixing of the liquid sample and the first, second andthird reagents occurs within first, second and third microfluidicchannels 520, 530 and 540.

In addition, similar to FIGS. 1A and 2A, first, second and thirdmicrofluidic channels 520, 530 and 540 may comprise one or more opticalviewing areas 560, 562 and 564 to enable visual verification that theliquid sample and the first, second and third reagents are flowingthrough first, second and third microfluidic channels 520, 530 and 540.In addition, optical viewing areas 560, 562 and 564 enable a user tovisually observe reactions occurring between the liquid same and thefirst, second and third reagents.

Microfluidic device 510 may be used as a rapid, disposable, blood typingassay. Such an assay may be utilized, for example, to provide bedsideconfirmation of a patient's ABO group prior to a blood transfusion.FIGS. 6A-6F are schematic illustrations of blood typing cards inaccordance with aspects of the present invention.

FIG. 6A illustrates a microfluidic device, or a card, 600. In thisembodiment reagent inlets for antibody-A 602, antibody-B 604, andantibody-D 606 are illustrated. Alternatively, as noted above withrespect to FIGS. 5A-5C, such reagents may be loaded during themanufacture of device 600 and inlets 602, 604 and 606 may be eliminatedby replacing such inlets with first, second and third blister pouchescontaining the reagents. For ease of use, inlets 602, 604 and 606, whichprovide access to filling the corresponding reservoirs 608, 610, and612, respectively, are optionally marked with decorative indicators 614,616, and 618.

FIG. 6A further shows a sample inlet 620 for accepting a blood sample orother fluid sample for testing. In the present embodiment sample 620 islabeled with a decorative indicator 622. The decorative indicator 622encircles a transparent window 624 that provides a visual indicator ofthe reservoir for the fluid accepted through sample inlet 620. Inalternative embodiments, window 624 may be omitted.

FIG. 6A further illustrates verification windows for the three reagents626, 628 and 630. These verification windows are aligned over thecorresponding microfluidic channels in order to provide visualverification that the reagents are in fact traveling through themicrofluidic channels as designed. As with the reagent inlets, thereagent verification windows are appropriately marked.

FIG. 6A further illustrates appropriately marked optical viewing areas632, 634 and 636 for viewing the blood typing results. In the currentembodiment a legend 638 is provided to interpret the visual results andaid the user in determining the blood type. A further legend 640 isprovided to aid the user in determining whether the blood is Rh positiveor Rh negative.

FIG. 6A further shows a bellows pump 642 for actuating fluid flowthrough the device. The bellows pump is fluidly connected with an outletport 644.

The embodiment in FIG. 6A further comprises an aperture 646 designed toaccept an affixing device such that the microfluidic device may beattached directly to the container of fluid or bag of blood to be bloodtyped. In alternate embodiments, the affixing mechanism may includeadhesive tape, a tie mechanism, a clamp, or may simply be inserted in apocket on the fluid container, or any other standard means of affixingthe device in position.

FIG. 6B illustrates an embodiment of microfluidic device 600 including afaceplate 650 attached to the device. FIG. 6B shows the inlets,verification windows, legends, and markings as shown in FIG. 6A,however, FIG. 6B further shows an open faceplate or cover plate 650attached to device 600. In the illustrated embodiment, faceplate 650 ishingedly connected to the device 600. In alternate embodiments, thefaceplate may be detached. When faceplate 650 is in an open position,the exposed side may further include operational instructions 652 forthe convenience of the user. The faceplate additionally protects theviewing windows and inlets of the device when device 600 is not in use.

FIG. 6C illustrates yet another embodiment and shows microfluidic device600 with a closed faceplate 650, covering the inlets, viewing windows,and legends shown in FIG. 6A, and a sheath 690. The sheath in thepresent embodiment is slideable and when slid in a downward direction, alower lip 692 of the sheath provides a locking mechanism holding thefaceplate in place. The faceplate 650, as noted previously, providesprotection to the underlying inlets, viewing windows, legends, andlegend drawings contained on the device. The faceplate 650 mayadditionally be used as a containment mechanism after the blood typingis complete, thus preventing contact with the blood or fluid beingtested. FIG. 6D further illustrates the embodiment of FIG. 6C and showsthe device when sheath 690 is slid into the locking position, thusholding faceplate 650 in the closed position.

FIG. 6E illustrates another embodiment of the attached faceplate 650. Inthis embodiment the faceplate 650 includes operational instructions 652for completing the blood typing test. The faceplate cover in thisembodiment further includes an adhesive strip 654 that may be used toseal the sample inlet, or alternatively may be used to hold thefaceplate closed. FIG. 6E further illustrates that the sheath 690 inthis embodiment is covering the antigen reservoirs. In a furtherembodiment, downward movement of the sheath 690 may be utilized toactuate release of the antigens from the reservoirs.

FIG. 6F shows an alternative configuration of device 600 and layout foruser ease. In FIG. 6F, the reagent verification windows 626, 628 and 630are grouped together for easier verification. Furthermore, in thisembodiment, a header 670 is included identifying the blood type windows.Further use of the legends on alternative embodiments may include use ofspecific colors to delineate various functions on the substrate. Forexample, a red circle may encircle the blood port.

FIGS. 7A-7C are a series of cross-sectional views of a microfluidicdevice 710 illustrating the operation of a sixth embodiment of theinvention. Microfluidic device 710 illustrated in FIG. 7A comprises afirst microfluidic channel 720 having a first end 722 and a second end724, a second microfluidic channel 730 having a first end 732 and asecond end 734, and a third microfluidic channel 740 having a first end742 and a second end 744. Sample inlet 718 is fluidly connected to firstends 722, 732 and 742 of first, second and third microfluidic channels720, 730 and 740.

Rather than comprising first, second and third reagent inlets forreceiving first, second and third reagents, similar to device 510 ofFIGS. 5A-5C, first microfluidic channel 720 of device 710 comprises afirst dried reagent zone 712 wherein a first reagent in printed, secondmicrofluidic channel 730 of device 710 comprises a second dried reagentzone 714 wherein a second reagent is printed, and third microfluidicchannel 740 comprises a third dried reagent zone 716 wherein a thirdreagent is printed. The first, second and third reagents may be printedonto first, second and third microfluidic channels 720, 730 and 740,respectively, during the manufacture of device 710 by methods such asink jet printing, micro drop printing and transfer printing.

As illustrated, bellows pump 750 is fluidly connected to second ends724, 734 and 744 of first, second and third microfluidic channels 720,730 and 740, and first, second and third liquid barriers 726, 736 and746 are interposed between bellows pump 750 and second ends 724, 734 and744 of first, second and third microfluidic channels 720, 730 and 740.As in FIGS. 1A, 2A, 3A and 5A, first, second and third liquid barriers726, 736 and 746 are gas permeable and liquid impermeable membranes.

As shown, bellows pump 750 is fluidly connected to a check valve 752,which permits fluid flow away from bellows pump 750. Alternatively, thebellows pump may comprise a vent hole as in the embodiments of FIGS. 1A,3A and 5A.

During operation, a liquid sample in placed into sample inlet 718,bellows pump 750 is depressed, either manually by a user or mechanicallyby an external device, and, then, bellows pump 750 is released. Duringdepression of bellows pump 750, check valve 752, or a vent hole (notshown), prevents fluid flow from bellows pump 750 into first, second andthird microfluidic channels 720, 730 and 740. Upon release of bellowspump 750, a negative fluid pressure is created in first, second andthird microfluidic channels 720, 730 and 740 and the liquid sample isdrawn into, and through, first, second and third microfluidic channels720, 730 and 740 to first, second and third liquid barriers 726, 736 and746 (as shown in FIG. 7C). As the liquid sample passes through first,second and third dried reagent zones 712, 714 and 716, the liquid samplehydrates the first, second and third reagents and mixing of the liquidsample and the first, second and third reagents occurs within first,second and third microfluidic channels 720, 730 and 740.

In addition, similar to FIGS. 1A, 2A and 5A, first, second and thirdmicrofluidic channels 720, 730 and 740 may comprise one or more opticalviewing areas 760, 762 and 764 to enable visual verification that theliquid sample and the first, second and third reagents are flowingthrough first, second and third microfluidic channels 720, 730 and 740.In addition, optical viewing areas 760, 762 and 764 enable a user tovisually observe reactions occurring between the liquid same and thefirst, second and third reagents.

FIGS. 8A-8C are a series of cross-sectional views of a microfluidicdevice 810 illustrating the operation of a seventh embodiment of theinvention. Microfluidic device 810 illustrated in FIG. 8A comprises afirst microfluidic channel 820 having a first end 822 and a second end824, a second microfluidic channel 830 having a first end 832 and asecond end 834, and a third microfluidic channel 840 having a first end842 and a second end 844. Sample inlet 818 is fluidly connected to firstends 822, 832 and 842 of first, second and third microfluidic channels820, 830 and 840.

Device 810 further comprises a first dried reagent zone 812 wherein afirst reagent in printed, a second dried reagent zone 814 wherein asecond reagent is printed, and a third dried reagent zone 816 wherein athird reagent is printed. The first, second and third reagents may beprinted during the manufacture of device 810 by methods such as ink jetprinting, micro drop printing and transfer printing. As illustrated,device 810 also comprises a hydrating buffer inlet 870 for receiving ahydrating buffer. In alternate embodiments, the hydrating buffer may beloaded during the manufacture of device 810 and hydrating buffer inlet870 may comprise, for example, a hydrating buffer blister pouch (notshown) containing the hydrating buffer. Such a blister pouch is adaptedto burst, or otherwise release the hydrating buffer into device 810,upon actuation, such as, for example, depression of the blister poucheither manually by a user or mechanically by an external device.

As illustrated, hydrating buffer inlet 870, and each of first driedreagent zone 812, second dried reagent zone 814, and third dried reagentzone 816 are fluidly connected to first ends 822, 832 and 842 of first,second and third microfluidic channels 820, 830 and 840. Bellows pump850 is fluidly connected to second ends 824, 834 and 844 of first,second and third microfluidic channels 820, 830 and 840, and first,second and third liquid barriers 826, 836 and 846 are interposed betweenbellows pump 850 and second ends 824, 834 and 844 of first, second andthird microfluidic channels 820, 830 and 840. First, second and thirdliquid barriers 826, 836 and 846 are gas permeable and liquidimpermeable membranes.

As shown, bellows pump 850 is fluidly connected to a check valve 852,which permits fluid flow away from bellows pump 780. Alternatively, thebellows pump may comprise a vent hole.

During operation, a liquid sample in placed into sample inlet 818 and ahydrating buffer is placed into hydrating buffer inlet 870. (In thealternate embodiment, wherein hydrating buffer inlet 870 comprises ahydrating buffer blister pouch containing the hydrating buffer,operating is commenced by placing a liquid sample into sample inlet 818and manually actuating the blister pouch to release the hydratingbuffer.) Bellows pump 850 is then depressed, either manually by a useror mechanically by an external device, and, then, bellows pump 850 isreleased. During depression of bellows pump 850, check valve 852, or avent hole (not shown), prevents fluid flow from bellows pump 850 intofirst, second and third microfluidic channels 820, 830 and 840. Uponrelease of bellows pump 850, a negative fluid pressure is created infirst, second and third microfluidic channels 820, 830 and 840 and theliquid sample and the hydrating buffer are drawn into, and through,first, second and third microfluidic channels 820, 830 and 840 to first,second and third liquid barriers 826, 836 and 846 (as shown in FIG. 8C).As the hydrating buffer passes through first, second and third driedreagent zones 812, 814 and 816, the hydrating buffer hydrates the first,second and third reagents and, subsequently, mixing of the liquid sampleand the first, second and third reagents occurs within first, second andthird microfluidic channels 820, 830 and 840.

In addition, similar to FIGS. 1A, 2A, 5A and 7A, first, second and thirdmicrofluidic channels 820, 830 and 840 may comprise one or more opticalviewing areas 860, 862 and 864 to enable visual verification that theliquid sample and the first, second and third reagents are flowingthrough first, second and third microfluidic channels 820, 830 and 840.In addition, optical viewing areas 860, 862 and 864 enable a user tovisually observe reactions occurring between the liquid same and thefirst, second and third reagents.

From the foregoing, and as set forth previously, it will be appreciatedthat, although specific embodiments of the invention have been describedherein for purposes of illustration, various modifications may be madewithout deviating from the spirit and scope of the invention. A personof ordinary skill in the art will appreciate that a plurality ofmicrofluidic channels, inlets, valves, membranes, pumps, liquid barriersand other elements may be arranged in various configurations inaccordance with the present invention to manipulate the flow of a fluidsample in order to prepare such sample for analysis. Accordingly, theinvention is not limited except as by the appended claims.

What is claimed is:
 1. A microfluidic device comprising: a firstmicrofluidic channel having a first end and a second end; a sample inletfluidly connected to the first end of the first microfluidic channel,the sample inlet configured to receive a liquid sample; an active valvepositioned between the sample inlet and the first end of the firstmicrofluidic channel; a first bellows pump fluidly connected to thesecond end of the first microfluidic channel; a liquid barrierpositioned between the first bellows pump and the second end of thefirst microfluidic channel, wherein the liquid barrier is gas permeableand liquid impermeable; a second microfluidic channel having a first endand a second end, wherein the first end of the second microfluidicchannel is fluidly connected to the first microfluidic channel at alocation adjacent to the active valve; a passive valve positionedbetween the first end of the second microfluidic channel and the firstmicrofluidic channel, wherein the passive valve is configured to openwhen fluid pressure in the first microfluidic channel is greater thanfluid pressure in the second microfluidic channel; and a samplereservoir fluidly connected to the second end of the second microfluidicchannel.
 2. The microfluidic device of claim 1, wherein the firstbellows pump comprises a vent hole.
 3. The microfluidic device of claim1, wherein the microfluidic device comprises a second bellows pumpconfigured to actuate the active valve.
 4. The microfluidic device ofclaim 1, wherein the sample reservoir comprises a vent hole.
 5. Amicrofluidic device comprising: first and second microfluidic channels,each of the first and second microfluidic channels having a first endand a second end; a sample inlet fluidly connected to the first end ofthe first microfluidic channel, the sample inlet configured to receivethe liquid sample; a first bellows pump fluidly connected to, andpositioned between, the second end of the first microfluidic channel andthe first end of the second microfluidic channel; a second bellows pumpfluidly connected to the second end of the second microfluidic channel,wherein the second bellows pump has a fluid outlet; a first check valvepositioned between the sample inlet and the first end of the firstmicrofluidic channel, wherein the first check valve is configured topermit fluid flow towards the first microfluidic channel; a second checkvalve positioned between the second end of the first microfluidicchannel and the first bellows pump, wherein the second check valve isconfigured to permit fluid flow towards the first bellows pump; a thirdcheck valve positioned between the first bellows pump and the first endof the second microfluidic channel, wherein the third check valve isconfigured to permits fluid flow towards the second microfluidicchannel; and a fourth check valve positioned between the second end ofthe second microfluidic channel and the second bellows pump, wherein thefourth check valve is configured to permit fluid flow towards the secondbellows pump.